Many of the procedures presently conducted in the field of nuclear medicine involve radiopharmaceuticals which provide diagnostic images of blood flow (perfusion) in the major organs and in tumors. The regional uptake of these radiopharmaceuticals within the organ of interest is proportional to flow; high flow regions will display the highest concentration of radiopharmaceutical, while regions of little or no flow have relatively low concentrations. Diagnostic images showing these regional differences are useful in identifying areas of poor perfusion, but do not provide metabolic information of the state of the tissue within the region of apparently low perfusion.
There is a need for new radiopharmaceuticals which specifically localize in hypoxic tissue, i.e., tissue which is deficient in oxygen, but still viable. These compounds should be retained in regions which are hypoxic, but should not be retained in regions which are normoxic. A radiopharmaceutical with these properties will display relatively high concentrations in such hypoxic regions, with low concentrations, in normoxic and infarcted regions. Diagnostic images with this radiopharmaceutical should readily allow the identification of tissue which is at risk of progressing to infarction, but still salvagable in, for example, the heart and brain.
It is well known that tumors often have regions within their mass which are hypoxic. These result when the rapid growth of the tumor is not matched by the extension of tumor vasculature. A radiopharmaceutical which localizes preferentially within regions of hypoxia could also be used to provide images which are useful in the diagnosis and management of therapy of tumors as suggested by Chapman, xe2x80x9cMeasurement of Tumor Hypoxia by Invasive and Non-Invasive Proceduresxe2x80x94A Review of Recent Clinical Studiesxe2x80x9d, Radiother. Oncol. 20(S1), 13-19 (1991). Additionally, a compound which localizes within the hypoxic region of tumors, but is labeled with a radionuclide with suitable xcex1- or xcex2-emissions could be used for the internal radiotherapy of tumors.
As reported by Martin et al. (xe2x80x9cEnhanced Binding of the Hypoxic Cell Marker [3E] Fluoro-misonidazolexe2x80x9d, J. Nucl. Med., Vol. 30, No. 2, 194-201 (1989)) and, Hoffman et al. (xe2x80x9cBinding of the Hypoxic Tracer [H-3] Misonidazole in Cerebral Ischemiaxe2x80x9d, Stroke, Vol. 18, 168 (1987) hypoxia-localizing moieties, for example, hypoxia-mediated nitroheterocyclic compounds (e.g., nitroimidazoles and derivatives thereof) are known to be retained in hypoxic tissue. In the brain or heart, hypoxia typically follows ischemic episodes produced by, for example, arterial occlusions or by a combination of increased demand and insufficient flow. Additionally, Koh et al., (xe2x80x9cHypoxia Imaging of Tumors Using [F-18]Fluoronitroimidazolexe2x80x9d, J. Nucl. Med., Vol. 30, 789 (1989)) have attempted diagnostic imaging of tumors using a nitroimidazole radiolabeled with 18F. A nitroimidazole labeled with 123I has been proposed by Biskupiak et al. (xe2x80x9cSynthesis of an (iodovinyl)misonidazole derivative for hypoxia imagingxe2x80x9d, J. Med. Chem., Vol. 34, No. 7, 2165-2168 (1991)) as a radiopharmaceutical suitable for use with single-photon imaging equipment.
While the precise mechanism for retention of hypoxia-localizing compounds is not known, it is believed that nitroheteroaromatic compounds, such as misonidazole, undergo intracellular enzymatic reduction (for example, J. D. Chapman, xe2x80x9cThe Detection and Measurement of Hypoxic Cells in Tumorsxe2x80x9d, Cancer, Vol. 54, 2441-2449 (1984)). This process is believed to be reversible in cells with a normal oxygen partial pressure, but in cells which are deficient in oxygen, further reduction can take place. This leads to the formation of reactive species which bind to or are trapped as intracellular components, providing for preferential entrapment in hypoxic cells. It is necessary, therefore, for hypoxia imaging compounds to possess certain specific properties; they must be able to traverse cell membranes, and they must be capable of being reduced, for example, by reductases such as xanthine oxidase.
The hypoxia imaging agents mentioned above are less than ideal for routine clinical use. For example, the positron-emitting isotopes (such as 18F) are cyclotron-produced and short-lived, thus requiring that isotope production, radiochemical synthesis and diagnostic imaging be performed at a single site or region. The costs of procedures based on positron-emitting isotopes are very high, and there are very few of these centers worldwide. While 123I-radiopharmaceuticals may be used with widely-available gamma camera imaging systems, 123I has a 13 hour half-life (which restricts the distribution of radiopharmaceuticals based on this isotope) and is expensive to produce. Nitroimidazoles labeled with 3H are not suitable for in vivo clinical imaging and can be used for basic research studies only.
The preferred radioisotope for medical imaging is 99mTc. Its 140 keV xcex3-photon is ideal for use with widely-available gamma cameras. It has a short (6 hour) half life, which is desirable when considering patient dosimetry. 99mTc is readily available at relatively low cost through commercially-produced 99Mo/99mTc generator systems. As a result, over 80% of all radionuclide imaging studies conducted worldwide utilize this radioisotope. To permit widespread use of a radio-pharmaceutical for hypoxia imaging, it is necessary that the compound be labeled with 99mTc. For radiotherapy, the rhenium radioisotopes, particularly 186Re and 188Re, have demonstrated utility.
EP 411,491 discloses boronic acid adducts of rhenium dioxime and technetium-99m dioxime complexes linked to various nitroimidazoles. Although these complexes are believed to be useful for diagnostic and therapeutic purposes, it would be desirable to obtain higher levels of the rhenium or technetium radionuclide in the targeted area, than are achieved with this class of capped-dioxime nitroimidazole complexes. It was demonstrated that the compounds disclosed in EP 411,491 possess reduction potentials similar to 2-nitroimidazole derivatives known to localize in hypoxic regions. In addition, the reduction of these compounds is catalyzed by xanthine oxidase. However, these compounds have poor membrane permeability. Thus, while these compounds might be retained by hypoxic cells, delivery of these compounds to the intracellular domain of these cells may be less than ideal. In addition, the complexes described in EP 411,491 require a heating step to form the hypoxia-localizing radiolabeled compounds. It would be more convenient for the routine use of such hypoxia-localizing radiolabeled compounds to be able to prepare such complexes at ambient temperatures.
Radiolabeled complexes of hypoxia-localizing moieties which retain the biochemical behavior and affinity of such moieties, which are labeled at room temperature with a suitable, easy-to-use radionuclide, and which are capable of providing increased amounts of the desired radionuclide to the targeted area, would be a useful addition to the art.
In accordance with the present invention, novel ligands, metal complexes of such ligands, processes for their preparation, and diagnostic and therapeutic methods for their use, are disclosed. In particular, metal complexes, e.g., technetium and rhenium complexes, which are linked toga hypoxia localizing moiety, and wherein the complex has a permeability through cell membranes greater than that of 14C-sucrose, are disclosed. Exemplary complexes are useful as diagnostic imaging agents in the case of technetium radionuclides and improved agents for radiotherapy in the case of rhenium radionuclides. Suitable novel ligands to form these complexes may include, but are not limited to di-, tri- or tetradentate ligands forming neutral complexes of technetium or rhenium with the metal preferably in the +5 oxidation state. Examples of such ligands are represented by the formulae 
where at least one R is xe2x80x94(A)pxe2x80x94R2 where (A)p is a linking group and R2 is a hypoxia localizing moiety; and wherein the other R groups are the same, or different and are independently selected from hydrogen, halogen, hydroxy, alkyl, alkenyl, alkynyl, alkoxy, aryl, xe2x80x94COOR3, 
xe2x80x94NH2, hydroxyalkyl, alkoxyalkyl, hydroxyaryl, haloalkyl, arylalkyl, -alkyl-COOR3, -alkyl-CON(R3)2, -alkyl-N(R3)2, -aryl-COOR3, -aryl-CON(R3)2, -aryl-N(R3)2, 5- or 6-membered nitrogen- or oxygen-containing heterocycle; or two R groups taken together with the one or more atoms to which they are attached form a carbocyclic or heterocyclic, saturated or unsaturated spiro or fused ring which may be substituted with R groups;
R1 is hydrogen, a thiol protecting group or xe2x80x94(A)pxe2x80x94R2;
R3 is hydrogen, alkyl or aryl;
m=2 to 5; and,
p=0 to 20.
It should be apparent that the disulfide of Ic can be reduced to the corresponding dithiol of Ib by known methodology prior to complexing with a metal.
The linking group (A)p can be any chemical moiety which can serve to physically distance, or otherwise isolate, the hypoxia localizing moiety from the rest of the complex of formula I. This might be important if the hypoxia localizing moiety is likely to be inhibited in its action by the rest of the complex. For example, in the linking group, wherein p is one, A, or the various A units in forming a straight or branched chain if p greater than 1, are independently selected from xe2x80x94CH2xe2x80x94, xe2x80x94CHR4xe2x80x94, xe2x80x94CR4R5xe2x80x94, xe2x80x94CHxe2x95x90CHxe2x80x94, xe2x80x94CHxe2x95x90CR4xe2x80x94, xe2x80x94CR4xe2x95x90CR5xe2x80x94, xe2x80x94Cxe2x89xa1Cxe2x80x94, cycloalkyl, cycloalkenyl, aryl, heterocyclo, oxygen, sulfur, 
wherein R4 and R5 are independently selected from alkyl, alkenyl, alkoxy, aryl, 5- or 6-membered nitrogen or oxygen containing heterocycle, halogen, hydroxy or hydroxyalkyl.
In considering the various linking groups known in the art, it is understood that p could be any convenient value depending upon the design choices for the desired complex. Preferably, p is xe2x89xa620 and most preferably pxe2x89xa610.
Listed below are definitions of the terms used to describe the complexes of this invention. These definitions apply to the terms as they are used throughout the specification (unless they are otherwise limited in specific instances) either individually or as part of a larger group.
The terms xe2x80x9calkylxe2x80x9d, xe2x80x9calkenylxe2x80x9d and xe2x80x9calkoxyxe2x80x9d refer to both straight and branched chain groups. Those groups having 1 to 10 carbon atoms are preferred.
The term xe2x80x9carylxe2x80x9d refers to phenyl and substituted phenyl. Preferred are phenyl and phenyl substituted with 1, 2 or 3 alkyl, haloalkyl, aminoalkyl, alkylaminoalkyl, dialkylaminoalkyl ,alkoxy, alkoxyalkyl, halogen, amino, hydroxy, or formyl groups.
The terms xe2x80x9chalidexe2x80x9d, xe2x80x9chaloxe2x80x9d and xe2x80x9chalogenxe2x80x9d refer to fluorine, chlorine, bromine and iodine.
The expression xe2x80x9c5- or 6-membered nitrogen containing heterocyclexe2x80x9d refers to all 5- and 6-membered rings containing at least one nitrogen atom. Exemplary aliphatic nitrogen heterocyclic derivatives have the formula 
wherein r is 0 or 1 and A is xe2x80x94Oxe2x80x94, xe2x80x94Nxe2x80x94R6, xe2x80x94Sxe2x80x94 or xe2x80x94CHxe2x80x94R6 wherein R6 is hydrogen, alkyl, aryl or arylalkyl. Such groups include pyrrolidinyl, piperidinyl, morpholinyl, piperazinyl, 4-alkyl-piperazinyl, 4-alkylpiperidinyl, and 3-alkyl-pyrrolidinyl groups. Also included within the expression xe2x80x9c5- or 6-membered nitrogen containing heterocyclexe2x80x9d are aromatic groups. Exemplary aromatic groups are pyrrolyl, imidazolyl, oxazolyl, pyrazolyl, pyridinyl, thiophenyl, pyridazinyl, thiazolyl, triazolyl and pyrimidinyl groups. The above groups can be linked via a hetero atom or a carbon atom.
The expression xe2x80x9c5- or 6-membered nitrogen or oxygen containing heterocyclexe2x80x9d refers to all 5- and 6-membered rings containing at least one nitrogen or oxygen atom. Exemplary groups are those described above under the definition of the expression xe2x80x9c5- or 6-membered nitrogen containing heterocyclexe2x80x9d. Additional exemplary groups are 1,4-dioxanyl and furanyl.
It has now been found that metal complexes having a permeability through cell membranes greater than that of 14C-sucrose provide enhanced products when linked to a hypoxia localizing moiety. Depending upon the metal used, complexes employing such hypoxia-localizing moiety-containing ligands are useful as imaging agents, therapeutic agents, radiosensitizers and hypoxic tissue cytotoxins.
Cell permeability is a property of a cell membrane which describes the mobility of extraneous molecules (permeants) within the internal structure of the membrane (W. D. Stein, xe2x80x9cTransport and Diffusion Across Cell Membranexe2x80x9d, New York Academic Press Inc. (1986); A. Kotyk, K. Janacek, J. Koryta, Biophysical Chemistry of Membrane Functions, Chichester, UK: John Wiley and Sons, (1988)). Molecules to which the membrane is permeable are able to penetrate through the membrane to reach the environment on the opposite side.
The examples which follow utilize a model of cell permeability based on the studies of Audus and Borchardt (xe2x80x9cBovine Brain Microvessel Endothelial Cell Monolayers as a Model system for the Blood-Brain Barrierxe2x80x9d, Ann. New York Accad. Sci., 1988; 9-18). The model consists of a cultured monolayer of bovine brain endothelial cells, which form tight intercellular junctions. Transit of compounds across the monolayer reflects the ability of such compounds to cross the intact cell membrane by passive, active and/or facilitated diffusion mechanisms. The rate of transit is compared with 3H2O (a highly permeable tracer) and 14C-sucrose (a non-permeable tracer). As discussed above, in accordance with the present invention, it has been found that complexes containing a hypoxia localizing moiety and having cell permeability greater than that of sucrose provide benefits to diagnostic and/or therapeutic procedures employing such complexes.
The present complexes, when used with a radioactive metal, provide levels of radionuclide within hypoxic tissue sufficient to enhance diagnostic and therapeutic methods employing such complexes.
Exemplary complexes of the present invention can be shown as 
where the R groups are as defined above, where M can be a radioactive or non-radioactive metal which may have other ligand(s) X and/or Y in the unfilled coordination sites. For example, in the cases where M=rhenium or technetium, the 
portion can be shown as 
Any radioactive metal can be employed in the present complexes, for example, technetium or rhenium for the complexes of Ibxe2x80x2, and technetium for the complexes of Iaxe2x80x2. Rhenium includes Re-186 and Re-188 radionuclides and mixtures thereof, and may also include Re-185 and Re-187. Technetium includes Tc-99m, Tc-94m and Tc-96.
Complexes of the present invention have not been heretofore disclosed and are useful in that they utilize the properties of the hypoxia localizing group to provide imaging or treatment of hypoxic tissue at a particular site. The complexes of the present invention wherein M is technetium provide highly effective, relatively easy to use diagnostic imaging products which are characterized by a covalent bond between the radionuclide complex and the hypoxia localizing group while substantially retaining the retention properties of the free hypoxia localizing group. It can be appreciated that typical examples of diagnostic uses for the complexes of the present invention when M is technetium include, but are not limited to, imaging of hypoxic tissue, present under pathological conditions in e.g., the heart, brain, lungs, liver, kidneys or in tumors.
In addition to being useful in imaging hypoxic tissue, the present complexes can also be used as blood flow markers, i.e., for perfusion imaging. The initial distribution of the novel complexes is proportional to blood flow and therefore imaging carried out soon after administration is an indicator of perfusion. A short time later, as the present complexes wash out of the normoxic tissue but are retained in the hypoxic tissue, imaging of the hypoxic tissue is realized.
Additionally, the present invention provides stably bound complexes when M is Re for radio-therapeutic indications. To the extent that hypoxic tissue is known to be present in tumors, Re complexes of the present invention are suitable for radiotherapy. The compounds of this invention when M is Re for use in radiotherapy can be injected into humans and concentrate in hypoxic tissue. This allows for the targeting of radionuclides to the desired sites with great specificity. It is understood, however, that radiotherapy will only be possible in those areas where a sufficient quantity of hypoxic tissue is present so as to provide therapeutic levels of rhenium to the area needing treatment.
Examples of hypoxia localizing groups are hypoxia-mediated nitro-heterocyclic groups, (i.e., nitro-heterocyclic groups that can be trapped by hypoxia-mediated reduction of the nitro moiety). In addition to those described in the Koh et al. and Hoffman et al. references above, hypoxia-localizing moieties may include those described in xe2x80x9cThe Metabolic Activation of Nitro-Heterocyclic Therapeutic Agentsxe2x80x9d, G. L. Kedderis et al., Drug Metabolism Reviews, 19(1), p. 33-62 (1988), xe2x80x9cHypoxia Mediated Nitro-Heterocyclic Drugs in the Radio- and Chemotherapy of Cancerxe2x80x9d, C. E. Adams, et al., Biochem. Pharmacology, Vol. 35, No. 1, pages 71-76 (1986); xe2x80x9cStructure-Activity Relationships of 1-Substituted 2-Nitroimidazoles: Effect of Partition Coefficient and Sidechain Hydroxyl Groups on Radiosensitization In vitroxe2x80x9d, D. M. Brown et al., Rad. Research, 90, 98-108 (1982); xe2x80x9cStructure-Activity Relationships in the Development of Hypoxic Cell Radiosensitizers.xe2x80x9d, G. E. Adams et al., Int. J. Radiat. Biol., Vol. 35, No. 2, 133-150 (1979); and xe2x80x9cStructure-Activity Relationships in the Development of Hypoxic Cell Radiosensitizersxe2x80x9d, G. E. Adams et al., Int. J. Radiat. Biol., Vol. 38, No. 6, 613-626 (1980). These all disclose various nitro-heterocyclic moieties suitable for incorporation into the complexes of the present invention and are incorporated herein by reference. These compounds comprise a nitro-heterocyclic group which may include a sidechain, (A)p, which can serve as the linking group connecting the nitro-heterocyclic portion to the rest of the complex of formula I of this invention.
When the hypoxia localizing group is a hypoxia-mediated nitro-heterocyclic group, the linker/localizing group portion of the complex can be represented by 
the ring portion being a 5- or 6-membered cyclic or aromatic ring, wherein
n is the total number of substitution positions available on the 5- or 6-membered ring;
the one or more R7 substituents are independently selected from hydrogen, halogen, hydroxy, alkyl, aryl, alkoxy, hydroxy-alkyl, hydroxyalkoxy, alkenyl, arylalkyl, arylalkylamide, alkylamide, alkylamine and (alkylamine)alkyl;
X1 can be nitrogen, oxygen, sulfur, xe2x80x94CR4, xe2x80x94CR7xe2x95x90, CR7R7 or xe2x80x94CRRxe2x80x94; and
when (A)p is absent (i.e., p=0) the nitro-heterocyclic hypoxia localizing moiety is linked to the rest of the complex of this invention via a nitrogen or carbon atom of the cyclic ring.
The references, above, regarding hypoxia localizing moieties serve to illustrate that the present thinking in the art is that the reduction potential of the nitro-heterocyclic group directly affects its retention in hypoxic tissue. The linking group, (A)p, may therefore be selected not only according to its capacity to distance the hypoxia localizing moiety from the rest of the complex, but also in accordance with its effect on the reduction potential of the hypoxia-mediated nitro-heterocyclic group.
Preferred hypoxia localizing moieties (shown with the linking groups) are 2-, 4- and 5-nitroimidazoles which can be represented by 
and nitrofuran and nitrothiazole derivatives, such as 
Exemplary groups (including (A)p linking groups) include, but are not limited to, 
where q=0 to 5. Most preferred are nitroimidazoles and derivatives thereof.
The ligands of formula Ia can be prepared by known methods such as those described in U.S. Pat. No. 4,615,876. For example an alkylene diamine of the formula 
is reacted with one equivalent of the chloro oxime 
to provide the diamine monooxime 
When the compound of formula Ia includes a hypoxia-localizing moiety (and optional linking group) on one but not both of the oxime portions, compound IV prepared as above, is reacted with 
to provide 
Alternatively, to prepare a compound of the formula Iaxe2x80x3, a compound of the formula IIIxe2x80x2 may be reacted with a compound of the formula II, and the diamine monoxime formed having the structure: 
reacted with a compound of the formula III.
Compounds of formula Ia having the hypoxia localizing moiety, R2, (and optional linking group) on the alkylene portion 
where s=0 to 4 and t=0 to 4 with the proviso that s+t is not greater than 4, can be prepared by reacting a compound of the formula 
with two equivalents of a compound of formula III when the oxime portions are to be identically substituted. Similarly, when the oxime portions are to include different substituents, a compound of formula V can be reacted with one equivalent of a first compound of formula III and the so-formed intermediate can thereafter be reacted with one equivalent of a second compound of formula IIIxe2x80x2.
Exemplary compounds of the formula Ia also include the disubstituted compounds: 
which may be prepared by reacting two equivalents of a compound of the formula IIIxe2x80x2 with one equivalent of a compound of the formula II; and the trisubstituted compounds 
which may be prepared by reacting two equivalents of a compound of the formula IIIxe2x80x2 with one equivalent of a compound of the formula V.
A novel and preferred process for preparing the compounds of formula Ia is outlined below. This novel process is also useful for preparing any alkylene diaminedioiime.
The novel process for the preparation of PnAO derivatives could easily be adapted to prepare compounds outside of the scope of this disclosure by those skilled in the art.
The novel process involves the use of a haloketone instead of the chloro oxime of compounds III and IIIxe2x80x2 shown above. Thus, in its broad aspects, the novel process involves the preparation of alkylene diaminedioximes by first reacting an alkylene diamine with two equivalents of a haloketone and converting the so-formed diketone to the corresponding alkylene diaminedioxime. Similarly, where different oxime portions are desired the alkylene diamine can be reacted with one equivalent of a first haloketone and then with one equivalent of a second haloketone. The so-formed unsymmetrical diketone is converted to the corresponding dioxime by known methodology as discussed above.
For example, the diamine II of the formula 
can be reacted with the haloketone 
where halogen can be Br, Cl, I, F, preferably Br, to provide the diketone 
Diketone VII can be converted to the corresponding dioxime product by known methods, e.g., treatment with o-trimethylsilyl hydroxylamine.
When each of the oxime portions of the final product are intended to be different, the novel method herein involves reacting a compound of formula II with a chloro oxime of formula III to provide the diamine monooxime of formula IV. The monooxime IV can thereafter be reacted with the differently substituted haloketone VI to provide the monoketone 
Monoketone VIII can be converted to the corresponding dioxime product by known methods as described above.
Alternatively, to provide unsymmetrical oximes the diamine of II can be reacted with one equivalent of a first haloketone of VI and the so-formed intermediate can thereafter be reacted with an equivalent of a second haloketone of VI.
Specifically regarding the novel process to prepare products of formula Ia, a diamine of formula V can be reacted with two equivalents of the haloketone VI to provide the diketone intermediates of the formula 
Diketone VIIxe2x80x2 can be converted to the corresponding dioxime by known methods as described above, to provide the corresponding products of formula Ia where the xe2x80x94(A)pxe2x80x94R2 group is on the alkylene portion of the ligand.
Unsymmetrical compounds of formula Ia can be prepared using such starting materials in the methodology described above, i.e., the sequential coupling of two dissimilar haloketones of VI to an alkylene diamine of II or V.
Similarly, a compound of formula IV can be reacted with a compound of the formula 
where Rxe2x80x2=R with the proviso that one of the Rxe2x80x2 groups must be xe2x80x94(A)pxe2x80x94R2, e.g., 
to provide, in the case using VIxe2x80x2a, the corresponding ketone-oxime 
(where one of the Rxe2x80x2 groups must be xe2x80x94(A)pxe2x80x94R2)
Ketone-oxime IX can be converted to the dioxime of Iaxe2x80x3 using known methodology as shown above.
To prepare the compounds of the formula 
a compound of the formula 
(prepared as described in WO 89 10759 to Mallinckrodt) can be coupled with a compound of the formula
Lxe2x80x94(A)pxe2x80x94R2xe2x80x83xe2x80x83(XI)
where L is a leaving group, e.g., halogen, to provide 
The tertiary amine disulfide of formula XII can thereafter be reduced to the desired dithiol product of formula Ibxe2x80x2 (where R1=H) using known disulfide reducing agents, e.g., tris(2-carboxy-ethyl)phosphine, dithiothreitol, and the like, as disclosed for example in the aforementioned WO 89 10759. Alternatively, the disulfide X can be reduced to the dithiol form prior to coupling with compound XI. In this case, standard sulfide protection should be employed prior to coupling with XI.
To prepare the compounds of the formula 
a compound of formula V can be reacted with a compound of the formula 
(prepared as described in Kung et al., xe2x80x9cSynthesis and Biodistribution of Neutral Lipid-soluble Tc-99m Complexes that Cross the Blood-Brain-Barrierxe2x80x9d, J. Nucl. Med., 25, 326-332 (1984)) to provide compounds of the formula 
Treatment of compound XIV with a reducing agent, e.g., sodium borohydride, provides intermediates of the formula 
which can be reduced to the corresponding disulfide products of Ibxe2x80x3 using known sulfide reducing agents as discussed above.
Compounds of the formula 
where Z and/or W are xe2x80x94(A)pxe2x80x94R2 and the other of Z and W can be R, can be prepared using known peptide coupling methodology. For example, a compound of the formula 
can be coupled with a compound of the formula 
to provide intermediates of the formula 
Intermediate XVIII can thereafter be coupled with a compound of the formula 
wherein Z and W are as defined above in formula Ibxe2x80x3xe2x80x2, to provide 
Reduction of compound XX, e.g., by treatment with borane, provides compounds of Ibxe2x80x3xe2x80x2 having the following structure 
In all of the above reactions described for preparing compounds of this invention, it should be readily apparent to those skilled in the art that sulfur groups, amine groups and ketone groups may need to be protected during the various reactions and that the so-protected resulting products can thereafter be deprotected by known techniques.
All of the examples and the process description below where M is rhenium involve the use of xe2x80x9ccarrier rheniumxe2x80x9d except as otherwise noted. The phrase xe2x80x9ccarrier rheniumxe2x80x9d means that the rhenium compounds used contain non-radioactive rhenium at concentrations of  greater than 10xe2x88x927 M.
Preparation of the complexes of this invention wherein M is rhenium can be accomplished using rhenium in the +5 or +7 oxidation state. Examples of compounds in which rhenium is in the Re(VII) state are NH4ReO4 or KReO4. Re(V) is available as, for example, [ReOCl4](NBu4), [ReOCl4](AsPh4), ReOCl3(PPh3)2 and as ReO2(pyridine)4⊕. Other rhenium reagents known to those skilled in the art can also be used.
Preparation of the complexes of this invention wherein M is technetium can best be accomplished using technetium in the form of the pertechnetate ion. For Tc-99m, the pertechnetate ion can best be obtained from commercially available technetium-99m parent-daughter generators; such technetium is in the +7 oxidation state. The generation of the pertechnetate ion using this type of generator is well known in the art, and is described in more detail in U.S. Pat. Nos. 3,369,121 and 3,920,995. These generators are usually eluted with saline solution and the pertechnetate ion is obtained as the sodium salt. Pertechnetate can also be prepared from cyclotron-produced radioactive technetium using procedures well known in the art.
The formation of the technetium complexes proceeds best if a mixture of pertechnetate ion in normal saline is mixed with the appropriate ligand containing at least one R group of the form xe2x80x94(A)pxe2x80x94R2 where (A)p is a linking group and R2 is a hypoxia-localizing moiety. An appropriate buffer or physiologically acceptable acid or base may be used to adjust the pH to a value suitable for labeling the ligand. This will vary dependent upon the nature of the ligand; for example, for ligands of type Ia, a pH in the range between xcx9c5.5 to xcx9c9.5 should be used, and preferably a pH value in the range 7.0-8.5. For ligands of the type IIb, a pH value in the range 3-8 should be used, with a pH of xcx9c6.0 being preferred. A source of reducing agent is then added to bring the pertechnetate down to the oxidation state of Tc(V) for chelation with the ligand. Stannous ion is the preferred reducing agent, and may be introduced in the form of a stannous salt such as stannous chloride, stannous fluoride, stannous tartrate, stannous diethylenetriamine pentaacetic acid or stannous citrate, but other suitable reducing agents are known in the art. The reaction is preferably run in an aqueous or aqueous/alcohol mixture, at or about room temperature, using a reaction time of about 1 minute to about 1 hour. The reducing agent should be present at a concentration of 5-50 xcexcg/ml. The ligand should optimally be present in a concentration of 0.5-2 mg/ml.
Alternatively, the technetium complexes of this invention can be prepared by ligand exchange. A labile Tc(V) complex is prepared by the reduction of TcO4xe2x88x92 in the presence of a ligand which forms a labile technetium complex, such as mannitol, the hydroxycarboxylate ligands glucoheptonate, gluconate, citrate, malate or tartrate at a pH value that is appropriate for the exchange ligand in question (usually 5-8). A reducing agent such as the stannous salts described above is added, which causes the formation of a labile reduced complex of Tc with the exchange ligand. This reduced Tc complex is then mixed with the ligand containing xe2x80x94(A)pxe2x80x94R2 at an appropriate pH value (as described above). The labile exchange ligand is displaced from the metal by the ligand containing the hypoxia-localizing moiety, thus forming the desired technetium complexes of this invention.
It is convenient to prepare the complexes of this invention at, or near, the site where they are to be used. A single, or multi-vial kit that contains all of the components needed to prepare the complexes of this invention (other than the Rhenium or Technetium ion) is an integral part of this invention.
A single-vial kit would contain ligand, a source of stannous salt, or other pharmaceutically acceptable reducing agent, and be appropriately buffered with pharmaceutically acceptable acid or base to adjust the pH to a value as indicated above. It is preferred that the kit contents be in the lyophilized form. Such a single vial kit may optionally contain exchange ligands such as glucoheptonate, gluconate, mannitol, malate, citric or tartaric acid and can also contain reaction modifiers, such as diethylenetriamine-pentaacetic acid or ethylenediamine tetraacetic acid. Additional additives, such as solubilizers (for example xcex1-, xcex2- or xcex3-cyclodextrin), antioxidants (for example ascorbic acid), fillers (for example, NaCl) may be necessary to improve the radiochemical purity and stability of the final product, or to aid in the production of the kit.
A multi-vial kit could contain, in one vial, he ingredients except pertechnetate that are required to form a labile Tc(V) complex as described above. The quantity and type of ligand, buffer pH and amount and type of reducing agent used would depend highly on the nature of the exchange complex to be formed. The proper conditions are well known to those that are skilled in the art. Pertechnetate is added to this vial, and after waiting an appropriate period of time, the contents of this vial are added to a second vial that contains a source of the ligand containing the hypoxia-localizing moiety, as well as buffers appropriate to adjust the pH to its optimal value. After a reaction time of about 1 to 60 minutes, the complexes of the present invention are formed. It is advantageous that the contents of both vials of this multi-vial kit be lyophilized. As described for the single vial kit, additional additives may be necessary to improve the radiochemical purity and stability of the final product, or to aid in the production of the kit.
Alternatively, the multi-vial kit may contain a source of ligand containing the hypoxia localizing moiety in one vial and a source of stannous ion in the second vial. Pertechnetate is added to the vial containing ligand, and then the contents of the second vial are added to initiate labeling. As above, the quantity and type of ligand, buffer pH and reducing agent used would depend on the nature of the hypoxia-localizing ligand and reducing agent used. Again, it is advantageous that the contents of both vials be lyophilized.
The complexes of this invention can be administered to a host by bolus or slow infusion intravenous injection. The amount injected will be determined by the desired uses, e.g. to produce a useful diagnostic image or a desired radiotherapeutic effect, as is known in the art.
Preferred complexes of this invention are those wherein the hypoxia localizing moiety is a hypoxia-mediated nitro-heterocyclic group. Most preferred are those wherein the hypoxia localizing moiety is 2-nitroimidazole or a derivative thereof.
In the complexes of the present invention the preferred values for (A)p are alkyl, oxa-alkyl, hydroxyalkyl, hydroxyalkoxy, alkenyl, arylalkyl, arylalkylamide, alkylamide, alkylamine and (alkyl-amine)alkyl.
The most Preferred values for (A)p are selected from 
wherein A3 and A3xe2x80x2 are the same of different alkyl.
Preferred complexes are 
where M1 is technetium and M2 is technetium or rhenium and wherin at least one R group is xe2x80x94(A)pxe2x80x94R2.
The following examples are specific embodiments of this invention.